Apocarotenoids (APOs) are a class of carotenoid oxidation products with high structural and functional diversity. Apart from serving as precursors of phytohormones, fungal pheromones and vitamin A, several APOs act as signaling molecules involved in stress response and growth as regulators in plants. To comprehensively profile plant APOs, we established an improved ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap MS) analytical platform. The improved APO analytical platform consists of an optimized sequential APO sample preparation and multiple UHPLC-MS detection methods and was successfully used to identify and quantify multiple subclasses of APOs from tomato fruits. By integrating ultrasound-assisted extraction, solid phase extraction, and chemical derivatization, the improved sequential APOs sample preparation facilitates the simultaneous preparation of different subclasses of APOs from plant materials. In addition, multiple UHPLC-MS detection methods enables high throughput analysis of APOs. Application of this analytical strategy can make important contributions to understanding the function of these compounds and significantly facilitate the elucidation of plant APO metabolism.